Certain Galpha proteins, the GTPase component of the GPCR signaling pathways, can form complexes with tubulin, a component of the cytoskeleton. Remarkably, tubulin appears to transactivate Galpha (transferring GTP directly from tubulin to Galpha, and, inversly, Gs-alpha binding to microtubule plus-ends activates the tubulin GTPase and increases microtubule dynamics. Continued association between tubulin and Gs-alpha appears to sequester Gs-alpha from active involvement in cellular signaling. Chronic treatment of cells with antidepressant drugs moves Gs-alpha into a membrane compartment where it is more associated with adenylyl cyclase and less associated with tubulin. Chronic treatment with some drugs of abuse may also alter membrane signaling compartments and the relationship between Gs-alpha and microtubule proteins. we have been seeking to understand the molecular basis for these poorly understood phenomena. Modeling studies suggest that the switch1 regions of Gs-alpha that are sensitive to GTP binding interact directly with the GTP-binding site of tubulin; mutations in Galpha that block transactivation of Galpha by tubulin map to this region as well. In order to obtain deeper structural insight into the interactions that regulate complex formation between Galpha proteins and tubulin, we propose here to more fully characterize the purified Gs-alpha/tubulin dimer complex and to determine the crystal structure of the heterotrimeric complex in different nucleotide-bound states. These structural studies will provide direct insight into a remarkable mechanism of GTPase regulation - mediated at both the protein-structural and cytoskeletal levels - but importantly, they will also provide the basis for subsequent development of small molecules targeted to the Gs-alpha/tubulin interface that may prove useful in therapy for depression and drug addiction or dependency. The work represents a synergistic fusion of our laboratories bridging biochemical, cell biological and structural biological approaches. It addresses directly a significant and novel hypothesis for a regulatory interaction that that may ultimately contribute to fundamental understanding of the molecular and cellular events involved in response to chronic drug treatment and of the biology of mood and dependency.